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Mitotic phosphorylation activates hepatoma-derived growth factor as a mitogen

Allen D Everett1, Jun Yang, Monzur Rahman

  • 1Department of Pediatrics, Cardiology Division, Johns Hopkins University, 600 N. Wolfe Street, Baltimore, MD 21287, USA. aeveret3@jhmi.edu

BMC Cell Biology
|April 15, 2011
PubMed
Abstract

Insights

Hepatoma-derived growth factor (HDGF) is phosphorylated at S103 during mitosis, a modification crucial for its mitogenic function in vascular smooth muscle cells. This phosphorylation is vital for cell cycle progression.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Hepatoma-derived growth factor (HDGF) is a known mitogen for various cell types.
  • Previous studies identified HDGF as a phosphoprotein using mass spectrometry but lacked functional validation.
  • The functional significance of HDGF post-translational modifications remained unclear.

Purpose of the Study:

  • To investigate the phosphorylation of HDGF in vascular smooth muscle cells (VSMC).
  • To determine the functional consequence of HDGF phosphorylation, particularly at specific serine residues.
  • To elucidate the role of HDGF phosphorylation in cell cycle regulation and proliferation.

Main Methods:

  • Primary mouse aortic VSMC were used to study HDGF phosphorylation.
  • Site-directed mutagenesis was employed to substitute serine residues (S103, S165, S202) with alanine.
  • A phospho-specific antibody against S103-phosphorylated HDGF was developed.
  • Flow cytometry (FACS) analysis was performed to assess cell cycle progression.
  • Cells were treated with nocodazole to induce mitotic arrest.

Main Results:

  • HDGF was confirmed to be phosphorylated in primary mouse aortic VSMC.
  • Substitution of S103, S165, and S202 with alanine reduced HDGF phosphorylation.
  • Mitosis-specific phosphorylation of HDGF at S103 was demonstrated using a specific antibody.
  • HDGF-S103A mutants exhibited impaired mitogenic activity and caused G2/M cell cycle arrest.
  • Cells expressing HDGF-S103D (an phosphomimetic mutant) showed normal cell cycle progression.
  • Nocodazole treatment significantly increased S103 phosphorylation in VSMC.

Conclusions:

  • HDGF is a phosphoprotein, with S103 phosphorylation being mitosis-related.
  • Phosphorylation of S103 is essential for HDGF's mitogenic function.
  • Cell cycle-regulated phosphorylation of HDGF likely plays a significant role in vascular cell proliferation.

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